Reactive Oxygen Species Modulator-1 (ROMO-1) Polymorphism Rs6060566 and The Risk of Myocardial Infarction in Slovenian Subjects With Type 2 Diabetes Mellitus

We aimed to examine the role of the rs6060566 polymorphism of the reactive oxygen species modulator-1 (ROMO-1) gene in the development of myocardial infarction (MI) in Caucasians with type 2 diabetes mellitus (T2DM). Methods A total of 1072 subjects with T2DM were enrolled in cross-sectional case-control study: 335 subjects with MI and 737 subjects without clinical signs of coronary artery disease (CAD). Genetic analysis of the rs6060566 polymorphism was performed in all subjects. To assess the degree of coronary artery obstruction, a subpopulation of 128 subjects with T2DM underwent coronary computed tomography (CT) angiography. Next, endarterectomy samples were obtained during myocardial revascularization from diffusely diseased coronary arteries in 40 cases, which were analysed for ROMO-1 expression according to their genotype.

angiography. Next, endarterectomy samples were obtained during myocardial revascularization from diffusely diseased coronary arteries in 40 cases, which were analysed for ROMO-1 expression according to their genotype.

Results
There were no statistically signi cant associations between different genotypes or alleles of the rs6060566 polymorphism and MI in subjects with T2DM. The carriers of the C allele of the ROMO-1 rs6060566 had a threefold increased likelihood of having coronary artery stenosis (AOR = 3.27, 95% CI 1. 16-9.20). Furthermore, the carriers of the C allele showed higher number of positive cells for ROMO-1 expression in endarterectomy samples of coronary arteries.

Conclusions
In accordance to our study, the rs6060566 polymorphism of the ROMO-1 gene is not the risk factor for MI in Caucasians with T2DM. However, we found that subjects carrying the C allele were at a 3.27-fold increased risk of developing severe CAD compared with those who had nonobstructive CAD. Moreover, The C allele carriers showed statistically higher number of cells positive for ROMO-1 compared with T allele carriers in coronary endarterectomy samples. Background Type 2 diabetes mellitus (T2DM) is a heterogeneous group of metabolic disorders that affects around 8% of world population and was thought to be responsible for 5 million deaths in 2015 worldwide [1]. In addition, is one of the major risk factors for coronary artery disease (CAD) and more than 40% of patients with acute coronary syndrome (ACS) have diabetes mellitus (DM) [2]. Coronary artery disease is characterized by atherosclerosis in coronary arteries and can be asymptomatic, whereas ACS usually presents with a symptom, such as unstable angina, and is frequently associated with myocardial infarction (MI) regardless of the presence of CAD [3]. Because of the proatherosclerotic, proin ammatory, and prothrombotic states associated with diabetes, diabetic patients with ACS are at high risk of subsequent cardiovascular events [4]. The 7-year risk of developing MI in diabetic patients was comparable to the risk of MI in nondiabetic patients who had a prior MI, which suggests that diabetes contributes signi cantly to the development of MI and can possibly be considered as a coronary heart disease (CHD) risk equivalent [5]. In a physiological system, the imbalance between antioxidant defence mechanism and reactive oxygen species (ROS) production leads to oxidative stress and subsequent pathological conditions [6]. Coronary vascular disease (CVD) occurs as a consequence of accelerated atherosclerosis, mainly driven by oxidative stress [7]. Numerous defence genes are involved in maintaining the balance between oxidant production and their removal by ROS-scavenger enzyme systems.
Reactive oxygen species modulator − 1 (ROMO-1) gene is located in chromosome 20q11.22 [8] and produces a small transmembrane protein located in the inner mitochondrial membrane. It is a unique nonselective cation channel that is suggested to be regulated in response to uctuation in free iron concentration and of its redox state [9]. Nevertheless, ROMO-1 is vital for normal mitochondrial morphology and function [10] and its activation induces ROS production in mitochondrial respiratory chain leading to oxidative stress and cell death [11]. Moreover, ROMO-1 is involved in cell proliferation [12], cell apoptosis [13], it is thought to have a role in replicative senescence [14] and in carcinogenesis and tumour progression tumour cells [15].
Up to now, however, only one study examined relationship between ROMO-1 polymorphism and diabetes vascular complication [16]. The aim of the present study was to assess the potential role of the ROMO-1 polymorphism rs6060566 in the development of MI in Slovenian subjects with T2DM. Furthermore, we also explored ROMO-1 expression in coronary endarterectomy specimens with immunohistochemically staining.

Subjects
In this retrospective cross-sectional case-control study, 1072 unrelated Caucasians with T2DM lasting at least 10 years were enrolled. Participants were divided into two study groups: 335 subjects with MI and 737 subjects with no history of CAD, no signs of ischemic changes on electrocardiogram and no ischemic changes during submaximal stress testing. Subjects were classi ed as having T2DM according to the current American Diabetes Association criteria [17]. The diagnosis of MI was made according to the established universal criteria [18]. Subjects with MI were included in the study 1 to 9 months after the acute event.

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All subjects enrolled in the study belonged to Caucasian ethnicity. After an informed consent for the participation in the study was obtained, a detailed interview was made including active smoking status and blood was drawn for biochemical analyses and genotyping. Body mass index (BMI) was calculated as weight in kilograms divided by the height in meters square.
Coronary computed tomography angiography Further, to assess the degree of coronary artery obstruction, a subpopulation of 128 asymptomatic subjects with T2DM underwent coronary computed tomography (CT) angiography for diagnostic purposes at International Centre for Cardiovascular Diseases MC Medicor, Izola, Slovenia. Non-invasive visualization of epicardial coronary artery tree and detection of stenosis was performed on dual source Dual energy CT scanner (Siemens, Germany). The acquisition and reading of the coronary CT angiograms was assessed by BC, a senior expert cardiac radiologist. Normal coronary arteries were de ned by the absence of obstructive or nonobstructive atherosclerotic plaque. Nonobstructive CAD was de ned by the presence of plaque occupying a cross-sectional area stenosis <50%. The severity of CAD was classi ed by the degree of stenosis of the cross-sectional area (<50%, ≥50%≤75% and >75%) and by the number of diseased vessels (score from 0 to 3; as 0 for no vessel disease (VD), 1 for single VD, 2 for double VD and 3 for triple VD). Angiographically diagnosed diseased left main coronary artery (LMCA) was scored as 1 with disregarding stenosis in any of two major branches: left anterior descending (LAD) or left circum ex (LCx). In addition, if LMCA was not affected by atherosclerosis, we assigned score 1 for each LAD or/and LCx, respectively. At last, diseased right coronary artery (RCA) was scored as 1.

Genotyping
Genomic DNA was extracted from 100 μl of whole blood using a Qiagen isolation kit. The rs6060566 polymorphism of the ROMO-1 gene was genotyped by KBioscience Ltd using their own novel uorescence-based competitive allele-speci c polymerase chain reaction (KASP) assay. Details of the method used can be found at http://www.kbioscience.co.uk/.

Immunohistochemistry
In the second part of the study, we included 40 subjects with T2DM with angina pectoris that had surgical myocardial revascularization. Coronary endarterectomy tissue samples were obtained during myocardial revascularization from diffusely diseased coronary arteries. With respect to different rs6060566 genotypes, relative expression for ROMO-1 in resected tissue samples was analysed by immunohistochemistry.
Sections from formalin-xed, para n-embedded tissue blocks of 40 coronary endarterectomy specimens were cut at a thickness of 5 µm. Immunohistochemistry was carried out using a VENTANA BenchMark Ultra Slide staining System (Roche Diagnostics). Endarterectomy specimens were stained with antibody Zeiss Group, Germany), two researchers PN and DP independently evaluated the slides and manually counted ROMO-1 positive cells at 400 magni cation. Numerical areal density of cells which were immunoreactive for ROMO-1 was calculated (the number of positive cells per mm 2 ) as described before [19].

Statistical analyses
Normally distributed continuous variables were expressed as means ± standard deviations, and as Additionally, all variables that showed signi cant differences by univariate analysis were put into a stepwise multiple logistic regression. A p-value of <0.05 was considered to be statistically signi cant. Statistical analysis was performed using the SPSS program version 19 (SPSS Inc. Chicago, IL).

Clinical characteristics and biochemical analysis
The clinical characteristics and biochemical analysis of the Slovenian subjects with T2DM are listed in Table 1. Cases (335 subjects with MI) had lower BMI, lesser waist circumference and better-controlled hypertension. Additionally, they had a higher total and LDL cholesterol, triglycerides, and lower HDL cholesterol. Moreover, cases had longer duration of T2DM. The two groups of subjects were well matched with regard to age, gender, fasting glucose, HbA1c, hsCRP level and concomitant history of cerebrovascular insult (CVI) or transitory ischaemic attack (TIA).  Association analyses Further, binary logistic regression analyses for different genetic models found no signi cant associations between different genotypes or alleles of the rs6060566 polymorphism and the risk of MI in Slovenian subjects with T2DM. Estimates of odds ratio (OR)s were adjusted (AOR)s (Table 3) for the variables (BMI, waist circumference, diastolic blood pressure, total cholesterol, HDL and LDL cholesterols, triglycerides, duration of DM in years) that were signi cant in the univariate analyses (Table 1). No signi cant differences in genotype and allele frequency distribution for the rs6060566 polymorphism were observed among subjects with different distribution and extent of CAD, which was de ned by coronary CT angiography (Table 4).
Coronary computed tomography angiography A total of 128 subjects with T2DM underwent coronary angiography (Fig. 1). A single vessel disease (1 VD) was observed in 22 (17%) subjects, two-vessel disease (2 VD) in 41 (32%) and three-vessel disease (3 VD) in 16 (13%) subjects. Further, 49 (38%) subjects had normal all major epicardial coronary arteries (LMCA, LAD, LCx and RCA) on CT angiograms (Fig. 1a). Moreover, 97 (76%) subjects had nonobstructive CAD (cross-sectional area stenosis of < 50%), in 28 (22%) subjects a cross-sectional area stenosis of ≥ 50%≤75% was detected while only 3 subjects (2%) had stenosis of > 75% (Fig. 1a). As can be seen in  Table 5. The dependent variables describing the severity of CAD were the number of diseased and extent of stenosis (none diseased vessel and stenosis < 50% were used as references, respectively). Independent variables included in the model were dominant genetic model (TT genotype was used as reference), age, gender, lipid parameters and duration of T2DM in years. We did not observe any interactions between dominant genetic model and CAD without adjustment for the possible confounders (Table 5). Nevertheless, when well-known CAD risk factors (age, gender, lipid parameters and duration of T2DM in years) were xed in the model the association between carriers of the [CC + CT] genotypes and ≥ 50%≤75% cross-sectional area stenosis became statistically signi cant (p = 0.025, multinomial logistic regression). The carriers of the C allele of the ROMO-1 rs6060566 had a threefold increased likelihood of having coronary artery stenosis (AOR = 3.27, 95% CI 1.16-9.20, Table 5).  1a, subjects with 2 VD (14/41, 34.1%) and nonobstructive CAD (11/97, 11.3%) suffered from nonfatal MI more often than other subjects in both comparative groups (number of diseased vessels and percentage of the cross-sectional area stenosis). Of note, there was a statistically signi cant difference (p = 0.0096, Pearson χ 2 test) in frequency distribution between subgroups with and without MI with regard to the extent of the CAD (Fig. 1a). In contrast, no difference (p = 0.283; Fischer s Exact test) was observed between subgroups with regard to the coronary cross-sectional area stenosis (Fig. 1a). Regarding the number of the involved vessels, a signi cantly higher frequency (p = 0.013; Pearson χ 2 test) of MI was found in subjects with 2 VD. Interestingly, subjects with two affected coronary arteries showed a 3.72 fold risk for MI (OR = 3.72, 95% CI 1.27-10.84, Fig. 1a). Coronary angiography revealed that of the 128 subjects more than 50% of them had developed CAD in LAD (Fig. 1b), while the remainder of the coronary arteries were spared of atherosclerotic disease more frequently. Atherosclerotic changes were noticed in LMCA in 39 subjects (30.5%), while a slightly higher percentage of atherosclerotic disease was seen in LCx and RCA (41.4%) (Fig. 1b). Furthermore, the relationship between presence or absence of CAD and coronary arteries was statistically signi cant (p = 0.0055, Pearson χ 2 test; Fig. 1b). As depicted in Fig. 1c, subjects with CAD (CAD+) in LMCA or LAD had about 3.5-fold higher risk of experiencing MI (p = 0.007 for LMCA and p = 0.01 for LAD, Pearson χ 2 test; respectively) compared with subjects without CAD (CAD-). However, in subjects with diseased LCx and RCA MI occurred more frequently than in subjects with disease-free coronary arteries, although the difference was not statistically signi cant (p = 1.0 for LCx and p = 0.9 for RCA, Pearson χ 2 test; respectively). Immunohistochemistry At the end of this study, the coronary artery segments, which were obtained by endarterectomy from subjects with advanced atherosclerosis, were examined with immunohistochemical staining. A statistically signi cantly higher numerical areal density of ROMO-1 positive cells was found in 17 subjects with the C allele (Fig. 2) in comparison with 23 subjects with ROMO-1 TT genotype (wild type) (835 ± 215/mm 2 versus 412 ± 153/mm 2 ; p < 0.001, Student s t test).

Discussion
Our study investigated the role of oxidative stress ROMO-1 gene polymorphism rs6060566 in Slovenian subjects with T2DM who experienced MI. We found no association of the rs6060566 polymorphism with MI. In contrast, we have identi ed a signi cant relationship between carriers of the [CC + CT] genotypes (under assumption of the dominant genetic model) and ≥ 50%≤75% cross-sectional area stenosis. Thus, we found that subjects carrying the C allele were at a 3.27-fold increased risk of developing severe CAD compared with those who had nonobstructive CAD.
Moreover, a signi cantly higher frequency of MI was found in subjects with 2 VD as compared to the subjects in whom CAD was not detected by CTA. At rst glance, it seems that the frequency of subjects with MI increased with the number of involved vessels. Our results are partly supported by in-depth study conducted on 12.594 diabetes patients by Gyldenkerne et al. [20] who found that the extent of CAD is a major risk factor for MI and death in patients with DM. We could not con rm this increased risk for MI in a group of subjects with 3 VD, most likely because of small sample size. Although it is known that diabetic patients suffer from more extensive CAD and hence higher incidence of multi-vessel CAD than non-diabetic subjects [21], in our research, 3 VD was diagnosed only in 16 out of 128 subjects who underwent coronary angiography. On the other hand, it is important to note, that all of them were diagnosed without atherosclerotic lesions in LMCA and none of them have suffered a MI. Furthermore, all 16 subjects underwent either percutaneous coronary intervention (PCI) or coronary artery bypass grafting surgery (CABG). We can recognize that appropriate myocardial revascularization strategy with optimal medical therapy appear to led to reduced incidence of MI in this small group of Slovenian subjects with 3 VD. Recently, a large-scale meta-analysis based on 18.224 patients with DM receiving PCI and CABG has demonstrated the superiority of CABG in reducing mortality, MI and need for repeat revascularization in patients with DM and complex CAD (including LMCA and/or multivessel disease) [22]. However, in our study we did not examine clinical outcomes of PCI or CABG in subjects with complex CAD.
In our study, subjects with CAD localized in LMCA and LAD had signi cantly higher occurrence of nonfatal MI compared to subjects without CAD in respective coronary arteries. They had about a 3.5-fold higher risk of MI. Our observations parallel with well-known facts that diabetes approximately doubles the risk of MI and death among patients with known CAD [23,24]. On the other hand, the current study shows that MI is less frequent in subjects with disease-free coronary arteries. Thus, in the absence of angiographically signi cant CAD, patients with diabetes treated with contemporary prophylactic therapy have the same risk of cardiovascular events as patients without diabetes [25]. Therefore, optimal medical therapy and appropriate selection of myocardial revascularization strategy is critical for patients with DM [26].
T2DM patients are strongly prone to atherothrombotic complications in coronary arteries as well as in other macro-and microvasculature. One of major contributors to susceptibility for atherothrombotic complications is genetic background [27]. Many studies [28,29] have shown association of different genetic loci and their polymorphisms with higher risk of atherosclerosis, CAD and MI. Numerous identi ed risk polymorphisms have no known pathophysiological function, but others are involved in in ammatory response, lipid function, transportation, endothelial dysfunction or oxidative stress regulation.
As oxidative stress is recognized as central pathogenic process in accelerated atherosclerosis in T2DM, various studies [7] investigated associations of oxidative stress genes (e.g. Nicotinamide Adenine Dinucleotide Phosphate (NADPH1) oxidase, Myeloperoxidase, Glutathione peroxidase 1, Glutathione Stransferase [30], NAD(P)H1: Quinone oxidoreductase (NQO1), Superoxide dismutase 1 and 2 [31], Thioredoxin reductase 2 [32], Uncoupling protein 2 [33], etc.) with micro-and macrovascular complications in T2DM. In general, studies yielded con icting results, some of this discrepancies could be attributed to different study populations, races, small sample sizes, study types (i.e. retrospective) and design. It should be noted that many association studies were conducted on one single common polymorphism and hence have not scanned for the gene-gene or gene-environment in uences on the risk for MI.
Oxidative stress occurs when ROS production exceeds the elimination capacity of antioxidant system. Most ROS are generated in mitochondrial respiratory chain [34] and although by-products, they are vital for normal processes, such as the maintenance of the vascular tone, cell adhesion, immune responses and cellular growth [35]. Increased expression of ROMO-1, rstly identi ed in tumour cells, increases ROS production [34], leading to oxidative stress and cell death [11]. Product of ROMO-1 is small transmembrane protein located in inner mitochondrial membrane that has been recently found to be a unique nonselective cation channel. Its function is suggested to be regulated in response to uctuation of free iron concentration and redox state of iron [9]. It is proposed that ROMO-1 activation causes recruitment of B-cell lymphoma-extra-large (Bcl-xL) protein to the outer mitochondrial membrane that in turn reduces its membrane potential, resulting in ROS production [13].
ROMO-1 is essential protein involved in several cell functions. It is vital for maintaining mitochondrial cristae shape [10,36] and its loss causes mitochondrial cytochrome c leakage, which is one of a key molecule involved in the intrinsic apoptotic pathway [36]. Its role in TNF-alpha-induced apoptosis was furthermore con rmed by Kim et al. [13].
Hwang et al. [11] showed that enforced ROMO-1 expression leads to massive cell death -necrosis -by excessive ROS production. It was shown that in physiological states ROMO-1 derived ROS were indispensable for the proliferation of both normal and cancer cells, respectively [12]. Moreover, Chung el al. [36] showed that ROMO-1 might be principal factor in deregulation of nuclear factor-κB and related pathways that contribute to tumour cell proliferation and invasion. It is also thought to have a role in replicative senescence [14]. Lung, colorectal cancer and gliomas [38] have been linked to ROMO-1. In nonsmall cell lung cancer ROMO-1 can serve as disease biomarker and is predictor of poor survival and malignant effusions in these patients [39,40]. In colorectal cancer, ROMO-1 expression predicts poor survival and higher invasiveness [15]. Its role is also being investigated concerning pulmonary and renal brosis [41], obstructive sleep apnoea syndrome [42] and Fanconi anaemia [43].
Further, according to our knowledge, this is the rst study investigating the expression of the ROMO-1 in coronary endarterectomy samples. The C allele carriers showed statistically higher number of cells positive for ROMO-1 compared with T allele carriers. This result corroborates the ndings by Petrovič et al. [16], in which greater ROMO-1 expression was found in brovascular membranes of subjects with microvascular complication of T2DM, namely proliferative diabetic retinopathy. We also assumed that ROMO-1 rs6060566 polymorphism might be associated with MI. However, we failed to show the presumable association in Slovenian subjects with T2DM. One possible explanation for our results might be that some of the controls had clinically silent CAD that we did not detect with our inclusion criteria, or had other macro-or microvascular complications that could also be induced by oxidative stress. Otherwise, subjects with MI had longer duration of DM and higher levels of HbA1c. It is generally believed that the relative risk for MI increases with any increase in glycaemia above the normal range [44]. On the other hand, cases had better managed blood pressure, which is probably a consequence of recent MI, thus recently optimized medical therapy with presumed better compliance.

Conclusion
In conclusion, in this study we did not observe any association between rs6060566 polymorphism of the ROMO-1 gene and the risk of MI in Caucasians with T2DM. However, we found that subjects carrying the C allele were at a 3.27-fold increased risk of developing severe CAD compared with those who had nonobstructive CAD. Moreover, The C allele carriers showed statistically higher number of cells positive for ROMO-1 compared with T allele carriers in coronary endarterectomy samples. Furthermore, it seems likely that the extent of CAD was a risk factor for MI in a subgroup of subjects in whom two-vessel disease was diagnosed with CT coronary angiography.
New opportunities for research activity should open up to explore the contribution of oxidative stress on perpetuating the atherosclerotic process in coronary artery system in subjects with T2DM. Prof. dr. Matjaž Jereb, MD; dr. Dušica Pleterski-Rigler, MD and Mr. Tone Žaklelj constituted the quorum for the ethical review of research. None of the members had any con ict of interest in the matter on which they voted. All study participants were fully informed of the research objectives, and those who agreed to participate signed an informed consent form.
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